A Synthetic Electronic Nanopore for DNA
Sequencing and Stochastic Sensing
Submitted to
The 2014 Summer NSF REU Program
Sponsored By
The National Science Foundation
Grant ID No.: DUE-0756921
College of Engineering and Applied Science
University of Cincinnati
Cincinnati, Ohio
Prepared By:
Mr. Aaron Choi, Chemical Engineering, University of Cincinnati
Mr. Davis Sneider, Biomedical Engineering, The George Washington
University
Mr. Saifuddin Aijaz, Computer Science, University of Cincinnati
Report Review By:
Margaret J. Kupferle, PhD, PE
REU Faculty Co-Mentor
Department of Biomedical, Chemical, and Environmental Engineering
University of Cincinnati
July 18, 2014
Abstract _____________________________________________________________________________________
Nanopores are present in daily life, channels that control ion transport along with
other substance concentrations, yet the importance and full practical use of these
structures is just now being tapped. These ion channels carry currents across which
can then be used to measure lengths of DNA and other molecules. In order to make
advances in modern medicine, it is essential to know exactly how DNA affects every
aspect of life. . At the University of Cincinnati, saline solutions were used, containing
ions which would allow the sensing and determination of which ions are present. In
addition to ions, the hydraphile nanopore, developed by Dr. George Gokel, can be
used to properly identify and sense norovirus. However, Dr. George Gokel’s
nanopore is believed to be 6-8 picometers, but by passing DNA 1-2 nanometers
wide through such a pore, researchers hope to verify the pore width to be wider,
and determine a better estimate.
Introduction________________________________________________________________________________
DNA sequencing is essential to understanding the code of life. There are numerous
ways to sequence DNA in this day and age such as with the shotgun method, the
Illumina dye sequencing, Sanger sequencing and 454 sequencing. Current
generation sequencing has advanced so far that methods like Illumina sequencing
can sequence up to 300 million unique reads per run. However, current methods
require a substantial amount of money and time. They require amplification of DNA
as well as other expensive materials. DNA sequencing can cost 10000 dollars and
take up to a week with our current technology. Next gen technologies aim to reduce
costs and increase efficiency.
With the present expense and time of current sequencing technologies, scientists
have discovered a cheaper more viable alternative in nanopores. Nanopores have
the ability to read long base pair chains with great speed. Additionally, nanopores
remove the cost of amplification procedures with their ability of single molecule
sensitivity. Despite the clear positives of such sequencing, drawbacks still are
present. These drawbacks are evident in pore structure and molecular
configuration. The configuration is vital to which ions are able to pass through,
making the proper configuration of the nanopore a crucial factor. Nanopore
sequencing is possible by applying an electric current across the molecular
configuration which in turn is given a specific resistance when DNA translocates
through the nanopore. As each individual base passes through, different electrical
signals are received, allowing the base-pair order to be determined.
In order to thread the DNA however, the pore must first be large enough for DNA to
fit through. Double-stranded DNA has a diameter around 2.6-2.9 nm. Singlestranded DNA in comparison to dsDNA has about half of the diameter which calls for
a pore of around 1-1.5 nm in diameter for ssDNA to fit through.
Nanopores are typically made up of proteins, however the nanopore used in this
experiment is a synthetic nanopore composed of hydraphiles. The hydraphiles are
composed of Lariat ethers and carbon chains, highlighted in the figure below.
Fig. 1
The organic compound encircled in the figure is one of the lariat ethers, each of
which have a diameter of about 1 nm. The hydraphile is 40 angstroms long in total,
with each of the carbon chains being about 15 angstroms long. This hydraphile has
been used in experiments before, one of which tested its usage as a possible cure for
cancer by upsetting the ion balance in the cancer cells and an experiment which
discovered this conformation of the hydraphile with information on the fluorescent
properties of the pore.2 Other experimentation included testing of other molecules
to use as the head groups, how short the optimal length of the hydraphile should be3,
and the hydraphile’s toxicity to cells.2
The hydraphile nanopore was created by Dr. George Gokel and its ability to
sequence and sense DNA isn’t fully tapped. The ability of this nanopore will be
tested by running multiple base pair lengths of DNA through the pore, collecting
data on the resistances caused by the passage as well as the dwell time of the
specific lengths. In addition to sequencing, sensing of molecules such as the
norovirus will aid in the detection of diseases and viruses, helping to treat and
eliminate them from the human body. The hydraphile nanopore is expected to be
able to run DNA through its core, as well as be a decent indicator for the norovirus.
Materials & Methods______________________________________________________________________
All chemicals were purchased from Sigma Aldrich unless otherwise stated. The
hydraphile nanopore was a generous gift from George Gokel. Hydraphile vesicles
were prepared with 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPHPC, Avanti
Polar Lipids, Alabaster, AL) at a concentration of 10mg/mL in a film rehydration
process. After rehydration, vesicles were extruded at 200nm and stored at -80oC
until use. Three buffers of 5mM HEPES and either 1M KCl, MgCl2, or NaCl at pH 7.8
and a seawater solution (National Center for Marine Algae and Biota, East Boothbay,
ME) were used for initial conductance measurements. A minimum of 100 data
points were taken per solution. For DNA passage data, 25ng of Fermentas NoLimits
DNA fragments (250, 500, 1000, and 2500 bp) were used (Thermo Scientific,
Pittsburgh, PA). A minimum of 200 data points were taken per length.
Bilayer lipid membrane (BLM) measurements were taken using the BCH-1 chamber
(Eastern Scientific LLC, Rockville, MD). Electrical measurements were taken using
the Axon Axopatch 200B and Axopatch Digidata (Molecular Devices, Carlsbad, CA)
and data collection and analysis were performed using Clampex and Clampfit
software. Voltages ranged from 50-100mV and membranes were painted using a
DPHPC-hexane solution.
Results and Discussion___________________________________________________________________
Out of the four buffers which were tested, only three were successful, giving data
showing that a current of ions was indeed able to flow through the hydraphile
nanopore. Out of the three buffers that were successful in allowing the ion current
to pass through, there was a varying effect of the buffer on the current. After
analyzing the data in Clampfit, the average jump for each nanopore opening was
determined. The KCl buffer has an average resistance of 1.76 nano siemens and the
NaCl buffer has an average resistance of 1.54 nano siemens.
The buffer that is used for DNA sequencing was determined by comparing their
resistances. Whichever buffer has the least standard deviation should be used due to
lower variance in measurements because the lower deviation allows the
researchers have a more reliable buffer which in turn allows them to have more
certainty in disturbances being blockages by DNA rather than instability in the
membrane due to the buffer. However, the KCl buffer was used due to greater
membrane stability during testing, which is vital to the passage of DNA through the
nanopore.
Fig. 2 [uses data from 4]
The estimated conductance per pore obtained from the KCL buffer solution data is
approximately 2.8 nano Siemens with a standard deviation of nearly 1.3 nano
Siemens. Using a correlation between conductance and nanopore size it is estimated
that the hydraphile nanopore is within a range of 2.1 to 3.1 nanometers in diameter
making it large enough to pass double stranded DNA, which is approximately 2
nanometers wide. In comparison, the protein nanopore GP10 is about 3.6
nanometers in diameter and can be used to pass double stranded DNA as well.
Fig. 3
Fig. 4
Other results as shown in the charts above obtained over the duration of
experimentation were dwell time ranges for various DNA strand lengths. 250, 500,
1000, and 2500 base pair lengths were run through the nanopore causing negative
resistances. The charts above show that the majority of DNA passage events occur in
between two to five seconds [fig. 3] and cause the pore to be 10 to 15% blocked [fig.
4] for DNA which is 250 base pairs long. The data obtained will be used to help
predict average dwell times for various lengths of DNA and RNA and will also be
used compare the resistance caused by single stranded DNA to the resistance
caused by double stranded DNA.
Acknowledgements________________________________________________________________________
We would like to thank Dr. David Wendell and Ms. Elizabeth Wurtzler for
their lab equipment and endless help in our research. We finally would like to thank
the NSF for our funding through grant no: DUE - 075692.
References__________________________________________________________________________________
Timp, W. (2010). "Nanopore Sequencing: Electrical Measurements of the Code of
Life." IEEE, 9(3), 281.
1
Negin, S., Smith, B. A., Unger, A., Leevy, W. M., and Gokel, G. W. (2013). "Hydraphiles:
A Rigorously Studied Class of Synthetic Channel Compounds with In Vivo Activity."
International Journal of Biomedical Imaging, 2013, 11.
2
Gokel, G. W. (2000). "Hydraphiles: design, synthesis and analysis of a family of
synthetic, cation-conducting channels." Chemical Communications(1), 1-9.
3
Wendell, D., Jing, P., Geng, J., Subramaniam, V., Lee, T. J., Montemagno, C., and Guo, P.
(2009). "Translocation of double-stranded DNA through membrane-adapted phi29
motor protein nanopores." Nat Nano, 4(11), 765-772.
4